Do the COVID-19 Crisis, Ageing and Climate Change Put Swiss Fiscal Sustainability at Risk?

The ongoing coronavirus pandemic crisis as well as demographic and climate change pose major challenges for public finances. This article deals with the implications of demographic trends in Switzerland, i.e. the progressive ageing of the population and its impact on the country's public finances in the long run. As the analysis shows, the brunt of the demographic burden is borne by the old-age pension scheme, health and long-term care. This article also addresses the financial ramifications of the COVID-19 crisis and shows the need for economic policy action over the longer term to ensure the sustainability of public finances in Switzerland. Furthermore, a qualitative assessment of climate change is included, as it constitutes an additional major long-term challenge for public finances.

Autofluorescence is a Reliable in vitro Marker of Cellular Senescence in Human Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSC) are used in cell therapies, however cellular senescence increases heterogeneity of cell populations and leads to uncertainty in therapies' outcomes. The determination of cellular senescence is time consuming and logistically intensive. Here, we propose the use of endogenous autofluorescence as real-time quantification of cellular senescence in human MSC, based on label-free flow cytometry analysis. We correlated cell autofluorescence to senescence using senescence-associated beta-galactosidase assay (SA-ß-Gal) with chromogenic (X-GAL) and fluorescent (C12FDG) substrates, gene expression of senescence markers (such as p16INK4A, p18INK4C, CCND2 and CDCA7) and telomere length. Autofluorescence was further correlated to MSC differentiation assays (adipogenesis, chondrogenesis and osteogenesis), MSC stemness markers (CD90/CD106) and cytokine secretion (IL-6 and MCP-1). Increased cell autofluorescence significantly correlated with increased SA-ß-Gal signal (both X-GAL and C12FDG substrates), cell volume and cell granularity, IL-6/MCP-1 secretion and with increased p16INK4A and CCND2 gene expression. Increased cell autofluorescence was negatively associated with the expression of the CD90/CD106 markers, osteogenic and chondrogenic differentiation potentials and p18INK4C and CDCA7 gene expression. Cell autofluorescence correlated neither with telomere length nor with adipogenic differentiation potential. We conclude that autofluorescence can be used as fast and non-invasive senescence assay for comparing MSC populations under controlled culture conditions.

Usefulness of Fat Suppression Magnetic Resonance Imaging of Osteoporotic Vertebral Fractures in Preventing Subsequent Fractures After Kyphoplasty.

BACKGROUND: Preoperative magnetic resonance imaging with fat suppression (FS-MRI) is useful to detect bone marrow edema in osteoporotic vertebral fractures (OVFs) and thus can improve diagnostic accuracy and influence surgical strategy for percutaneous augmentation. The role of preoperative FS-MRI in preventing subsequent fractures after balloon kyphoplasty has not been investigated in initially subclinical fractures or fractures without obvious morphologic changes. METHODS: From January 2010 to December 2017, 214 consecutive patients underwent balloon kyphoplasty for painful OVFs. We defined 2 groups based on preoperative imaging (100 patients had preoperative FS-MRI and 114 patients had no MRI) and then compared baseline and surgical characteristics. The primary end point was incidence of subsequent fractures within 12 months after treatment. RESULTS: The 214 patients underwent kyphoplasty of 414 vertebrae. Comparing FS-MRI with no-MRI groups, spontaneous fractures occurred significantly more (58% vs. 26.3%; P < 0.001) and fractures were more often multilevel (≥ 4 levels) (15% vs. 2.6%; P = 0.001), respectively. Overall incidence of subsequent vertebral fractures was 25.7% (32% in FS-MRI, 20.2% in no-MRI groups; P = 0.048). Average time to diagnosis of subsequent fractures did not differ between the 2 groups (9.3 FS-MRI vs. 11.5 weeks no-MRI; P = 0.411). Age ≥80 years at the time of balloon kyphoplasty was associated with a higher odds ratio (2.3) for subsequent fractures within 12 months (P = 0.039). CONCLUSIONS: Surgical treatment according to preoperative FS-MRI did not reduce occurrence of subsequent OVFs and did not prolong fracture-free intervals within 12 months after kyphoplasty.

VEGF vascularization pathway in human intervertebral disc does not change during the disc degeneration process.

OBJECTIVE: During degeneration of the intervertebral disc ingrowth of blood vessels and nerves into the disc are associated with back pain. Vascular endothelial growth factors promote vasculogenesis by binding to the membrane vascular endothelial growth factor receptor 1, while shorter soluble forms of this receptor can inhibit vascularization. We hypothesized that membrane and soluble receptor forms might change between stages of intervertebral disc degeneration. RESULTS: Expression of soluble and membrane forms of vascular endothelial growth factor receptor 1 in human degenerated intervertebral discs and healthy bovine caudal discs was assessed by qRT-PCR and immunoblot. Comparative microarray meta-analysis across disc degeneration grades showed that membrane and soluble forms of this receptor, together with other components of classic vascularization pathways, are constitutively expressed across human disc degeneration stages. Contrary to our hypothesis, we observed that expression of the classic vascularization pathway is stable across degeneration stages and we assume that soluble vascular endothelial growth factor receptor 1 does not contribute to prevent disc degeneration. However, we observed increased expression levels of genes involved in alternative vascularization signalling pathways in severely degenerated discs, suggesting that abnormal vascularization is part of the pathological progression of disc degeneration.

Oxidative status predicts quality in human mesenchymal stem cells.

BACKGROUND: Human bone marrow-derived mesenchymal stem cells (MSC) are adult progenitor cells with great potential for application in cell-based therapies. From a cell-based therapy perspective, there are two limitations to MSC use: (1) these therapies require large numbers of cells, and long-term expansion of MSC in vitro promotes replicative senescence; and (2) patient variability is a challenge for defining MSC quality standards for transplantation. This study aimed to determine whether low or high oxidative status of MSC correlate with changes in cell expansion and differentiation potentials. METHODS: We investigated functional aspects of mitochondria, such as cell metabolic activity indicators and expression of antioxidant enzymes. Furthermore, we tested if senescence-induced changes in oxidative status of MSC could be counteracted by methylene blue (MB), an alternative mitochondrial electron transfer known to enhance cell bioenergetics. RESULTS: MSC isolated from donors of the same age showed distinctive behavior in culture and were grouped as weak (low colony-forming units (CFU) and a short life in vitro) and vigorous MSC (high CFU and a long life in vitro). In comparison to weak MSC, vigorous MSC had oxidative status characterized by lower mitochondrial membrane potential, lower mitochondrial activity, and fewer reactive oxygen species production, as well as reduced mitochondrial biogenesis. Vigorous MSC had a significantly higher expansion potential compared to weak MSC, while no differences were observed during differentiation. MB treatment significantly improved expansion and differentiation potential, however only in vigorous MSC. CONCLUSIONS: Together, these results demonstrate the importance of mitochondrial function in MSC in vitro, and that cells with low oxidative status levels are better candidates for cell-based therapies.

Increased motility of mesenchymal stem cells is correlated with inhibition of stimulated peripheral blood mononuclear cells in vitro.

Immunomodulatory properties of mesenchymal stem cells (MSC) are key components of their successful applications in clinical setting. However, treatments based on MSC immunomodulation need understanding of cell characteristics before cell transplantation. We used live-imaging to test the suitability of the MSC motility as a parameter for quick prediction of the immunomodulatory potential of human MSC in regulating the activity of stimulated peripheral blood mononuclear cells (PBMC) in vitro. Bone marrow MSC, from various donors and in vitro passages, were cultured with or without stimulated PBMC. After seven days, immunomodulation was assessed by measuring PBMC proliferation, IgG production and cytokine secretion in MSC and PBMC monocultures and co-cultures, and results were correlated to MSC motility. In co-culture, we observed that MSC successfully inhibited PBMC activity, reducing PBMC proliferation and IgG production compared to PBMC monoculture. MSC modulated PBMC to reduce the secretion of TNFα and IL-10, increase IL-6, G-CSF and MCP-1, while GM-CSF was not affected. By live-imaging tracking of cell trajectories, we observed that fast moving MSC were inhibiting more efficiently stimulated PBMC compared to slow ones. In co-culture, fast MSC were more effective in inhibiting IgG production (˜30% less IgG), and secreted higher levels of IL-10 (˜10% increase) and GM-CSF (˜20% increase) compared to slower cells. Furthermore, fast MSC in monocultures produced 2.3-fold more IL-6, 1.5-fold MCP-1 and 1.2-fold G-CSF in comparison to slower cells. In conclusion, live-imaging cell tracking allowed us to develop an indicative assay of the immune-regulatory potential of MSC prior to in vivo administration. Key Words: Human mesenchymal stem cells, Immunomodulatory potential, In vitro cell motility, Stem cell transplantation.

An in vitro expansion score for tissue-engineering applications with human bone marrow-derived mesenchymal stem cells.

Human bone marrow-derived mesenchymal stem cells (MSCs) have limited growth potential in vitro and cease to divide due to replicative senescence, which from a tissue-engineering perspective has practical implications, such as defining the correct starting points for differentiation and transplantation. Time spent in culture before the loss of required differentiation potential is different and reflects patient variability, which is a problem for cell expansion. This study aimed to develop a score set which can be used to quantify the senescent state of MSCs and predict whether cells preserve their ability to differentiate to osteogenic, adipogenic and chondrogenic phenotypes, based on colony-forming unit (CFU) assay, population doubling time (PDT), senescence-associated ß-galactosidase (SA-ß-Gal) activity, cell size, telomere length and gene expression of MSCs cultured in vitro over 11 passages. This set of morphological, physiological and genetic senescence markers was correlated to the ability of MSCs to differentiate. Differentiation efficiency was assessed by marker genes and protein expression. CFUs decreased with increasing passage number, whereas SA-ß-Gal activity and PDT increased; however, the correlation with MSCs' differentiation potential was sometimes unexpected. The expression of genes related to senescence was higher in late-passage cells than in early-passage cells. Early-passage cells underwent efficient osteogenic differentiation, with mid-passage cells performing best in chondrogenic differentiation. Late-passage cells preserve only adipogenic differentiation potential. Based on this marker set, we propose a senescence score in which combined markers give a reliable quality control of MSCs, not depending only on mechanistic passage number.

Growth Factors Cross-Linked to Collagen Microcarriers Promote Expansion and Chondrogenic Differentiation of Human Mesenchymal Stem Cells.

Tissue engineering is a field in progressive expansion and requires constant updates in methods and devices. One of the central fields is the development of biocompatible, biodegradable, and injectable scaffolds, such as collagen microcarriers. To enhance cell attachment and produce a cost-effective cell culture solution with local stimulation of cells, basic fibroblast growth factor (bFGF) or transforming growth factor-ß1 (TGF-ß1) was covalently immobilized on microcarriers either by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) or riboflavin/UV (RB/UV) light-mediated cross-linking. Collagen microcarriers cross-linked with bFGF or TGF-ß1 were used for expansion and chondrogenic differentiation of human mesenchymal stem cells (MSCs). Evaluation methods included cell viability test, chondrogenic marker expression (aggrecan and collagen type I and type II), histological detection of proteoglycans, and immunohistochemical analysis. Cross-linking strengthened the collagen structure of the microcarriers and reduced collagenase-mediated degradation. MSCs effectively proliferated on microcarriers cross-linked with bFGF, especially by EDC/NHS cross-linking. Chondrogenic differentiation of MSCs was induced by TGF-ß1 cross-linked on microcarriers, promoting gene expression and protein accumulation of aggrecan and collagen type I and type II, as well as proteoglycans. Cross-linking by RB/UV enhanced chondrogenesis more than any other group. In addition, cross-linking reduced scaffold shrinkage exerted by MSCs during chondrogenesis, a desirable feature for microcarriers if used as tissue defect filler. In conclusion, cross-linking of bFGF or TGF-ß1 to collagen microcarriers supported in vitro proliferation and chondrogenesis, respectively. If translated in vivo and in clinical practice, such approach might lead a step closer to development of a cost-effective and locally acting device for cell-based therapy.

An enzymatic method to rescue mesenchymal stem cells from clotted bone marrow samples.

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug - clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

In vitro cell motility as a potential mesenchymal stem cell marker for multipotency.

Mesenchymal stem cells (MSCs) are expected to have a fundamental role in future cell-based therapies because of their high proliferative ability, multilineage potential, and immunomodulatory properties. Autologous transplantations have the "elephant in the room" problem of wide donor variability, reflected by variability in MSC quality and characteristics, leading to uncertain outcomes in the use of these cells. We propose life imaging as a tool to characterize populations of human MSCs. Bone marrow MSCs from various donors and in vitro passages were evaluated for their in vitro motility, and the distances were correlated to the adipogenic, chondrogenic, and osteogenic differentiation potentials and the levels of senescence and cell size. Using life-image measuring of track lengths of 70 cells per population for a period of 24 hours, we observed that slow-moving cells had the higher proportion of senescent cells compared with fast ones. Larger cells moved less than smaller ones, and spindle-shaped cells had an average speed. Both fast cells and slow cells were characterized by a low differentiation potential, and average-moving cells were more effective in undergoing all three lineage differentiations. Furthermore, heterogeneity in single cell motility within a population correlated with the average-moving cells, and fast- and slow-moving cells tended toward homogeneity (i.e., a monotonous moving pattern). In conclusion, in vitro cell motility might be a useful tool to quickly characterize and distinguish the MSC population's differentiation potential before additional use.

Five-year results of lumbar disc prostheses in the SWISSspine registry.

PURPOSE: The Swiss Federal Office of Public Health demanded a nationwide HTA registry for lumbar total disc arthroplasty (TDA), to decide about its reimbursement. The goal of the SWISS spine registry is to generate evidence about the safety and efficiency of lumbar TDA. METHODS: Two hundred forty-eight cases treated between 3-2005 and 6-2006, who were eligible for the 5-year follow-up were included in the study. Follow-up rates for 3-6 months, 1, 2 and 5 years were 85.9, 77.0, 44.0 and 51.2 %, respectively. Outcome measures were back and leg pain, medication consumption, quality of life, intraoperative and postoperative complication and revision rates. Additionally, segmental mobility, ossification, adjacent and distant segment degeneration were analysed at the 5-year follow-up. RESULTS: There was a significant, clinically relevant and lasting reduction of back (preop/postop 73/29 VAS points) and leg pain (preop/postop VAS 55/22) and a consequently decreased analgesics consumption and quality of life improvement (preop/postop 0.30/0.76 EQ-5D score points) until 5 years after surgery. The rates for intraoperative and early postoperative complications were 4.4 and 3.2 %, respectively. The overall complication rate during five postoperative years was 23.4 %, and the adjacent segment degeneration rate was 10.7 %. In 4.4 % of patients, a revision surgery was performed. Cumulative survivorship probability for a revision/re-intervention-free 5-year postoperative course was 90.4 %. At the 5-year follow-up, the average range of motion of the mobile segments (86.8 %) was 9.7°. In 43.9 % of patients, osteophytes at least potentially affecting the range of motion were seen. CONCLUSIONS: Lumbar TDA appeared as efficient in long-term pain alleviation, consequent reduction of pain medication consumption and improvement of quality of life. The procedure also appeared sufficiently safe, but surgeons have to be aware of a list of potential adverse events. The outcome is stable over the 5-year postoperative period. The vast majority of treated segments remained mobile after 5 years, although almost half of patients showed osteophytes.

Physiological testosterone levels enhance chondrogenic extracellular matrix synthesis by male intervertebral disc cells in vitro, but not by mesenchymal stem cells.

BACKGROUND CONTEXT: Testosterone (T) is a hormone and regulator involved in the processes of development of the organism (ie, promoting development of bone and muscle mass). Although T effects on the mesenchyme-derived muscle, bone, and adipose tissues are well studied, T effects on intervertebral disc (IVD) have not been reported. PURPOSE: The aim was to test the following hypothesis: if a physiological concentration of T (∼30 nM) can improve in vitro chondrogenesis of human IVD cells and mesenchymal stem cells (MSCs). STUDY DESIGN/SETTING: Human IVD cells and MSCs were differentiated to chondrogenic lineage on gelatin scaffolds for 4 weeks, in the presence or absence of T. METHODS: Chondrogenesis was assessed by cell viability, by measuring gene expression with quantitative polymerase chain reaction and extracellular matrix (ECM) accumulation with immunoblotting, immunohistochemical, and biochemical methods. RESULTS: Supplementation of T to chondrogenic culture did not affect viability. In male IVD cells, T had a beneficial impact on chondrogenesis, particularly in nucleus pulposus cells, demonstrated by an increased expression of aggrecan, collagen type I, and especially collagen type II. Conversely, T had no effects on chondrogenesis of female IVD cells or MSCs from both genders. A gene expression array of transforming growth factor ß/bone morphogenetic protein signaling cascade showed that in male IVD cells, T promoted a stable general but nonsignificant increase in gene expression. Furthermore, aromatase inhibitor anastrazole repressed the effect of T on ECM expression by IVD cells. The results suggest that T increased ECM accumulation in male IVD cells in combination with its conversion to estradiol by the enzyme aromatase. CONCLUSIONS: We demonstrated that T effectively enhances in vitro chondrogenesis in male IVD cells, rising the interest in the possible role of sex hormones in IVD degeneration. Nevertheless, T does not affect chondrogenic differentiation of female IVD cells and MSCs from both genders.

Influence of different commercial scaffolds on the in vitro differentiation of human mesenchymal stem cells to nucleus pulposus-like cells.

INTRODUCTION: Cell-based therapies for regeneration of the degenerated intervertebral disc (IVD) are an alternative to current surgical intervention. Mesenchymal stem cells (MSCs), in combination with a scaffold, might be ideal candidates for regenerating nucleus pulposus (NP), the pressure-distributing part of the IVD. While the use of growth factors for MSCs differentiation currently receives major attention, in this study we compare the performance of sponge-like matrixes in supporting cell differentiation into NP-like cells. MATERIALS AND METHODS: Four types matrixes approved as medical devices for other applications were tested as scaffolds for MSCs: two made of equine or porcine collagen, one of gelatin and one of chitosan. Bone marrow-derived human MSCs were seeded in these scaffolds or embedded in alginate, as a three-dimensional control. After five weeks in culture, NP-like differentiation of the cell-scaffold constructs was analyzed by qRT-PCR, histology, total DNA quantification, proteoglycan accumulation and immunohistochemistry. RESULTS: MSCs in collagen matrixes and gelatin produced more mRNA and proteins of the chondrogenic markers collagen type I, collagen type II (COL2) and aggrecan (ACAN), when compared with cells embedded in alginate or chitosan. Proteoglycan accumulation and cell survival were also higher in collagen and gelatin matrixes. Gene expression results were also confirmed by histological and immunohistochemical staining. In contrast to alginate control, the gene expression of the undesired bone marker osteopontin was lower in all tested groups. In porcine collagen supports, MSC expression ratio between COL2/ACAN closely resembled the expression of nucleus pulposus cells, but gene expression of recently described NP markers keratin19, PAX1 and FOXF1 was lower. CONCLUSIONS: Collagen supports provide a readily available, medically approved and effective scaffold for chondrogenic differentiation in vitro, but the phenotype of differentiated MSCs is not yet completely equivalent to that of NP cells.

The in vitro effects of dexamethasone, insulin and triiodothyronine on degenerative human intervertebral disc cells under normoxic and hypoxic conditions.

Degeneration of intervertebral discs (IVD) is one of the main causes of back pain and tissue engineering has been proposed as a treatment. Tissue engineering requires the use of highly expensive growth factors, which might, in addition, lack regulatory approval for human use. In an effort to find readily available differentiation factors, we tested three molecules--dexamethasone, triiodothyronine (T3) and insulin--on human IVD cells isolated after surgery, expanded in vitro and transferred into alginate beads. Triplicates containing 40 ng/ml dexamethasone, 10 nM T3 and 10 µg/ml insulin, together with a positive control (10 ng/mL transforming growth factor (TGF)-beta 1), were sampled weekly over six weeks and compared to a negative control. Furthermore, we compared the results to cultures with optimized chondrogenic media and under hypoxic condition (2% O2). Glycosaminoglycan (GAG) determination by Alcian Blue assay and histological staining showed dexamethasone to be more effective than T3 and insulin, but less than TGF-beta1. DNA quantification showed that only dexamethasone stimulated cell proliferation. qPCR demonstrated that TGF-beta1 and the optimized chondrogenic groups increased the expression of collagen type II, while aggrecan was stimulated in cultures containing dexamethasone. Hypoxia increased GAG accumulation, collagen type II and aggrecan expression, but had no effect on or even lowered cell number. In conclusion, dexamethasone is a valuable and cost-effective molecule for chondrogenic and viability induction of IVD cells under normoxic and hypoxic conditions, while insulin and T3 did not show significant differences.

Human mesenchymal stem cell co-culture modulates the immunological properties of human intervertebral disc tissue fragments in vitro.

The capacity of mesenchymal stem cells (MSCs) to differentiate into intervertebral disc (IVD)-like cells has been well described, but their ability to modulate the inflammatory processes in the IVD remains unclear. We found that tissue obtained by discectomy of degenerated and post-traumatic IVD contains significant amounts of IgG antibodies, a sign of lymphocyte infiltration. Further we investigated whether MSCs in vitro, which were characterized for their multilineage differentiation potential and may have immunomodulatory effects on IVD fragments. IVD fragments were co-cultured in contact with peripheral blood lymphocytes (PBLs) and MSCs, and as functional controls we used contact co-cultures of PBLs stimulated with pokeweed mitogen (2.5 µg/mL) and MSCs. The time course of lymphocyte proliferation (Alamar Blue), IgG (ELISA) and gene expression (RT-PCR) of anti-inflammatory cytokines (TGF-ß1, IL-10) by MSCs and pro-inflammatory molecules (IL-1α, IL-1ß and TNF-α) by the IVD fragments were analyzed. Depending on the response to the presence of MSCs, the IVD fragments (n = 13) were divided in two groups: responders (n = 9), where inflammation was inhibited by MSCs and non-responders (n = 4), where MSCs did not decrease inflammation. At 1 week in co-culture, MSCs reduced significantly the IgG production in the IVD responders group to 69% and PBLs proliferation to 57% of the control. MSCs expression of the anti-inflammatory TGF-ß1 increased with time, while IL-10 was expressed only at day 1. IVD gene expression of TNF-α decreased constantly, whereas IL-1α and IL-1ß expression increased. In conclusion, these data suggest that MSCs may modulate disc-specific inflammatory and pain status and aid regeneration of the host tissue.

Phenocopy--a strategy to qualify chemical compounds during hit-to-lead and/or lead optimization.

A phenocopy is defined as an environmentally induced phenotype of one individual which is identical to the genotype-determined phenotype of another individual. The phenocopy phenomenon has been translated to the drug discovery process as phenotypes produced by the treatment of biological systems with new chemical entities (NCE) may resemble environmentally induced phenotypic modifications. Various new chemical entities exerting inhibition of the kinase activity of Transforming Growth Factor ß Receptor I (TGF-ßR1) were qualified by high-throughput RNA expression profiling. This chemical genomics approach resulted in a precise time-dependent insight to the TGF-ß biology and allowed furthermore a comprehensive analysis of each NCE's off-target effects. The evaluation of off-target effects by the phenocopy approach allows a more accurate and integrated view on optimized compounds, supplementing classical biological evaluation parameters such as potency and selectivity. It has therefore the potential to become a novel method for ranking compounds during various drug discovery phases.